The ability of the body to control the flow of blood following vascular injury is paramount to continued survival. The process of blood clotting and then the subsequent dissolution of the clot, following repair of the injured tissue, is termed hemostasis. Hemostasis is composed of a number of events that occur in a set order following the loss of vascular integrity:
The initial phase of the process is vascular constriction. This limits the flow of blood to the area of injury. Next, platelets become activated by thrombin and aggregate at the site of injury, forming a temporary, loose platelet plug. The protein fibrinogen is primarily responsible for stimulating platelet clumping. Platelets clump by binding to collagen that becomes exposed following rupture of the endothelial lining of vessels. Upon activation, platelets release adenosine-5′-diphosphate, ADP and TXA2 (which activate additional platelets), serotonin, phospholipids, lipoproteins, and other proteins important for the coagulation cascade. In addition to induced secretion, activated platelets change their shape to accommodate the formation of the plug.
To insure stability of the initially loose platelet plug, a fibrin mesh (also called the clot) forms and entraps the plug. Finally, the clot must be dissolved in order for normal blood flow to resume following tissue repair. The dissolution of the clot occurs through the action of plasmin.
Two pathways lead to the formation of a fibrin clot: the intrinsic and extrinsic pathway. Although they are initiated by distinct mechanisms, the two converge on a common pathway that leads to clot formation. The formation of a red thrombus or a clot in response to an abnormal vessel wall in the absence of tissue injury is the result of the intrinsic pathway. Fibrin clot formation in response to tissue injury is the result of the extrinsic pathway. Both pathways are complex and involve numerous different proteins termed clotting factors
Platelet Activation and Von Willebrand Factor (vWF).
In order for hemostasis to occur, platelets must adhere to exposed collagen, release the contents of their granules, and aggregate. The adhesion of platelets to the collagen exposed on endothelial cell surfaces is mediated by von Willebrand factor (vWF). The function of vWF is to act as a bridge between a specific glycoprotein on the surface of platelets (GPIb/IX) and collagen fibrils. In addition to its role as a bridge between platelets and exposed collagen on endothelial surfaces, vWF binds to and stabilizes coagulation factor VIII. Binding of factor VIII by vWF is required for normal survival of factor VIII in the circulation.
Von Willebrand factor is a complex multimeric glycoprotein that is produced by and stored in the platelets. It is also synthesized by megakaryocytes and found associated with subendothelial connective tissue. The initial activation of platelets is induced by thrombin binding to specific receptors on the surface of platelets, thereby initiating a signal transduction cascade. The thrombin receptor is coupled to a G-protein that, in turn, activates phospholipase C-γ (PLC-γ). PLC-γ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) leading to the formation of inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of intracellular Ca2+ stores, and DAG activates protein kinase C (PKC).
The collagen to which platelets adhere as well as the release of intracellular Ca2+ leads to the activation of phospholipase A2 (PLA2), which then hydrolyzes membrane phospholipids, leading to liberation of arachidonic acid. The arachidonic acid release leads to an increase in the production and subsequent release of thromboxane A2 (TXA2). This is another platelet activator that functions through the PLC-γ pathway. Another enzyme activated by the released intracellular Ca2+ stores is myosin light chain kinase (MLCK). Activated MLCK phosphorylates the light chain of myosin which then interacts with actin, resulting in altered platelet morphology and motility.
One of the many effects of PKC is the phosphorylation and activation of a specific 47,000-Dalton platelet protein. This activated protein induces the release of platelet granule contents; one of which is ADP. ADP further stimulates platelets increasing the overall activation cascade; it also modifies the platelet membrane in such a way as to allow fibrinogen to adhere to the platelet surface, resulting in fibrinogen-induced platelet aggregation.
Activation of platelets is required for their consequent aggregation to a platelet plug. However, equally significant is the role of activated platelet surface phospholipids in the activation of the coagulation cascade.
The intrinsic clotting cascade is initiated when contact is made between blood and exposed endothelial cell surfaces. The extrinsic and intrinsic pathways converge at the point where factor X is activated to factor Xa. Factor Xa has a role in the further activation of factor VII to Vila. Active factor Xa also hydrolyzes and activates prothrombin to thrombin. Thrombin can then activate factors XI, VIII and V furthering the cascade. Ultimately the role of thrombin is to convert fribrinogen to fibrin and to activate factor XIII to XIIIa. Factor XIIIa (also termed transglutaminase) cross-links fibrin polymers solidifying the clot.
The intrinsic pathway requires the clotting factors VIII, IX, X, XI, and XII. Also required are the proteins prekallikrein and high-molecular-weight kininogen, as well as calcium ions and phospholipids secreted from platelets. Each of these pathway constituents leads to the conversion of factor X (inactive) to factor Xa (active). Initiation of the intrinsic pathway occurs when prekallikrein, high-molecular-weight kininogen, factor XI and factor XII are exposed to a negatively charged surface. This is termed the contact phase. Exposure of collagen to a vessel surface is the primary stimulus for the contact phase.
The assemblage of contact phase components results in conversion of prekallikrein to kallikrein, which in turn activates factor XII to factor XIIa. Factor XIIa can then hydrolyze more prekallikrein to kallikrein, establishing a reciprocal activation cascade. Factor XIIa also activates factor XI to factor XIa and leads to the release of bradykinin, a potent vasodilator, from high-molecular-weight kininogen.
In the presence of Ca2+, factor XIa activates factor IX to factor IXa. Factor IX is a proenzyme that contains vitamin K-dependent γ-carboxyglutamate (gla) residues, whose serine protease activity is activated following Ca2+ binding to these gla residues. Several of the serine proteases of the cascade (II, VII, IX, and X) are gla-containing proenzymes. Active factor IXa cleaves factor X at an internal arg-ile bond leading to its activation to factor Xa.
The activation of factor Xa requires assemblage of the tenase complex (Ca2+ and factors VIIIa, IXa and X) on the surface of activated platelets. One of the responses of platelets to activation is the presentation of phosphatidylserine and phosphatidylinositol on their surfaces. The exposure of these phospholipids allows the tenase complex to form. The role of factor VIII in this process is to act as a receptor, in the form of factor VIIIa, for factors IXa and X. Factor VIIIa is termed a cofactor in the clotting cascade. The activation of factor VIII to factor VIIIa (the actual receptor) occurs in the presence of minute quantities of thrombin. As the concentration of thrombin increases, factor VIIIa is ultimately cleaved by thrombin and inactivated. This dual action of thrombin, upon factor VIII, acts to limit the extent of tenase complex formation and thus the extent of the coagulation cascade.
As discussed supra activated factor Xa is the site at which the intrinsic and extrinsic coagulation cascades converge. The extrinsic pathway is initiated at the site of injury in response to the release of tissue factor (factor III). Tissue factor is a cofactor in the factor Vila-catalyzed activation of factor X. Factor Vila, a gla residue containing serine protease, cleaves factor X to factor Xa in a manner identical to that of factor IXa of the intrinsic pathway. The activation of factor VII occurs through the action of thrombin or factor Xa. The ability of factor Xa to activate factor VII creates a link between the intrinsic and extrinsic pathways. An additional link between the two pathways exists through the ability of tissue factor and factor Vila to activate factor IX. While there is some uncertainty it appears the formation of complex between factor Vila and tissue factor is believed to be a principal step in the overall clotting cascade. A major mechanism for the inhibition of the extrinsic pathway occurs at the tissue factor-factor VIIa-Ca2+-Xa complex. The protein, lipoprotein-associated coagulation inhibitor, LACI specifically binds to this complex. LACI is also referred to as extrinsic pathway inhibitor, EPI or tissue factor pathway inhibitor, TFPI and was formerly named anticonvertin. LACI is composed of 3 tandem protease inhibitor domains. Domain 1 binds to factor Xa and domain 2 binds to factor Vila only in the presence of factor Xa
Activation of Prothrombin to Thrombin
The common point in both extrinsic and intrinsic pathways is the activation of factor X to factor Xa. Factor Xa activates prothrombin (factor II) to thrombin (factor IIa). Thrombin, in turn, converts fibrinogen to fibrin. The activation of thrombin occurs on the surface of activated platelets and requires formation of a prothrombinase complex. This complex is composed of the platelet phospholipids, phosphatidylinositol and phosphatidylserine, Ca2+, factors Va and Xa, and prothrombin. Factor V is a cofactor in the formation of the prothrombinase complex, similar to the role of factor VIII in tenase complex formation. Like factor VIII activation, factor V is activated to factor Va by means of minute amounts and is inactivated by increased levels of thrombin. Factor Va binds to specific receptors on the surfaces of activated platelets and forms a complex with prothrombin and factor Xa.
Prothrombin is a 72,000-Dalton, single-chain protein containing ten gla residues in its N-terminal region. Within the prothrombinase complex, prothrombin is cleaved at 2 sites by factor Xa. This cleavage generates a 2-chain active thrombin molecule containing an A and a B chain which are held together by a single disulfide bond.
In addition to its role in activation of fibrin clot formation, thrombin plays an important regulatory role in coagulation. Thrombin combines with thrombomodulin present on endothelial cell surfaces forming a complex that converts protein C to protein Ca. The cofactor protein S and protein Ca degrade factors Va and VIIIa, thereby limiting the activity of these two factors in the coagulation cascade.
Thrombin also binds to and leads to the release of G-protein-coupled protease activated receptors (PARs), specifically PAR-1, -3 and -4. The release of these proteins leads to the activation of numerous signaling cascades that in turn increase release of the interleukins, ILs, IL-1 and IL-6, increases secretion of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). The thrombin-induced signaling also leads to increased platelet activation and leukocyte adhesion. Thrombin also activates thrombin-activatable fibrinolysis inhibitor (TAFI) thus modulating fibrinolysis (degradation of fibrin clots). TAFI is also known as carboxypeptidase U (CPU) whose activity leads to removal of C-terminal lysines from partially degraded fibrin. This leads to an impairment of plasminogen activation, thereby reducing the rate of fibrin clot dissolution (i.e. fibrinolysis).
Control of Thrombin Levels
The inability of the body to control the circulating level of active thrombin would lead to dire consequences. There are two principal mechanisms by which thrombin activity is regulated. The predominant form of thrombin in the circulation is the inactive prothrombin, whose activation requires the pathways of proenzyme activation described above for the coagulation cascade. At each step in the cascade, feedback mechanisms regulate the balance between active and inactive enzymes.
The activation of thrombin is also regulated by four specific thrombin inhibitors. Antithrombin III is the most important since it can also inhibit the activities of factors IXa, Xa, XIa and XIIa. The activity of antithrombin III is potentiated in the presence of heparin by the following means: heparin binds to a specific site on antithrombin III, producing an altered conformation of the protein, and the new conformation has a higher affinity for thrombin as well as its other substrates. This effect of heparin is the basis for its clinical use as an anticoagulant. The naturally occurring heparin activator of antithrombin III is present as heparin and heparin sulfate on the surface of vessel endothelial cells. It is this feature that controls the activation of the intrinsic coagulation cascade.
However, thrombin activity is also inhibited by α2-macroglobulin, heparin cofactor II and α1-antitrypsin. Although a minor player in thrombin regulation α1-antitrypsin is the primary serine protease inhibitor of human plasma. Its physiological significance is demonstrated by the fact that lack of this protein plays a causative role in the development of emphysema.
Activation of Fibrinogen to Fibrin
Fibrinogen (factor I) consists of 3 pairs of polypeptides ([A-α][B-β][γ])2. The 6 chains are covalently linked near their N-terminals through disulfide bonds. The A and B portions of the A-α and B-β chains comprise the fibrinopeptides, A and B, respectively. The fibrinopeptide regions of fibrinogen contain several glutamate and aspatate residues imparting a high negative charge to this region and aid in the solubility of fibrinogen in plasma. Active thrombin is a serine protease that hydrolyses fibrinogen at four arg-gly bonds between the fibrinopeptide and the a and b portions of the protein.
Thrombin-mediated release of the fibrinopeptides generates fibrin monomers with a subunit structure (α-β-γ)2. These monomers spontaneously aggregate in a regular array, forming a somewhat weak fibrin clot. In addition to fibrin activation, thrombin converts factor XIII to factor XIIIa, a highly specific transglutaminase that introduces cross-links composed of covalent bonds between the amide nitrogen of glutamines and e-amino group of lysines in the fibrin monomers.
Dissolution of Fibrin Clots
Degradation of fibrin clots is the function of plasmin, a serine protease that circulates as the inactive proenzyme, plasminogen. Any free circulating plasmin is rapidly inhibited by α2-antiplasmin. Plasminogen binds to both fibrinogen and fibrin, thereby being incorporated into a clot as it is formed. Tissue plasminogen activator (tPA) and, to a lesser degree, urokinase are serine proteases which convert plasminogen to plasmin. Inactive tPA is released from vascular endothelial cells following injury; it binds to fibrin and is consequently activated. Urokinase is produced as the precursor, prourokinase by epithelial cells lining excretory ducts. The role of urokinase is to activate the dissolution of fibrin clots that may be deposited in these ducts.
Active tPA cleaves plasminogen to plasmin which then digests the fibrin; the result is soluble degradation product to which neither plasmin nor plasminogen can bind. Following the release of plasminogen and plasmin they are rapidly inactivated by their respective inhibitors. The inhibition of tPA activity results from binding to specific inhibitory proteins. At least four distinct inhibitors have been identified, of which 2-plasminogen activator-inhibitors type 1 (PAI-1) and type 2 (PAI-2) are of greatest physiological significance.
Thus, from the above it can be seen that the physiological mechanisms involved in coagulation are exceedingly complex, and it will be appreciated that great difficulty exists in designing or identifying agents that are capable of safely modulating the many inter-related pathways in coagulation. The multilevel cascade of blood clotting system permits enormous amplification of its triggering signals. Moving down the extrinsic pathway, for example, proconvertin (VII), Stuart factor (X), prothrombin, and fibrinogen are present in plasma in concentrations of <1, 8, 150, and ˜4000 mg·mL-1, respectively. Thus a small signal is very quickly amplified to bring about effective hemostatic control.
Clotting must be very strictly regulated because even one inappropriate clot can have fatal consequences. Indeed, blood clots are the leading cause of strokes and heart attack, the two major causes of human death. Thus, the control of clotting is a major medical concern. Several inhibitors have been developed with different mechanisms of anticoagulant action. These include the heparins, the coumarins, and the 1,3-indanediones.
Heparin is a mucopolysaccharide with a molecular weight ranging from 6,000 to 40,000 Da. The average molecular of most commercial heparin preparations is in the range of 12,000-15,000. The polymeric chain is composed of repeating disaccharide unit of D-glucosamine and uronic acid linked by interglycosidic bonds. The uronic acid residue could be either D-glucuronic acid or L-iduronic acid. Few hydroxyl groups on each of these monosaccharide residues may be sulfated giving rise to a polymer with that is highly negatively charged. The average negative charge of individual saccharide residues is about 2.3.
The key structural unit of heparin is a unique pentasaccharide sequence. This sequence consists of three D-glucosamine and two uronic acid residues. The central D-glucosamine residue contains a unique 3-O-sulfate moiety that is rare outside of this sequence.
Heparin forms a high-affinity complex with antithrombin. The formation of antithrombin—heparin complex greatly increases the rate of inhibition of two principle procoagulant proteases, factor Xa and thrombin. The normally slow rate of inhibition of both these enzymes (˜103-104 M-1 s-1) by antithrombin alone is increased about a 1.000-fold by heparin. Accelerated inactivation of both the active forms of proteases prevents the subsequent conversion of fibrinogen to fibrin that is crucial for clot formation.
Heparin is relatively non-toxic, however heparin overdose or hypersensitivity may result in excessive bleeding. Protamines, are used as anti-dote for excessive bleeding complications.
Coumarin and its derivatives are principal oral anticoagulants. Warfarin is a coumarin derivative marketed as a racemic mixture of R and S isomers.
Coumarins are slow to act, exerting their effect in vivo only after a latent period of 12 to 4 hours and their effect lasts for 1.5 to 5 days. The observed slow onset may be due to the time required to decrease predrug prothrombin blood levels, whereas the long duration of action observed with warfarin may be due to the lag time required for the liver to resynthesize prothrombin to predrug blood levels.
Coumarins and 1,3-indandiones (see infra) have a further disadvantage in that they interact with certain drugs. For example, the action of oral anticoagulants can be enhanced by drugs such as phyenylbutazone and salicylates while antagonized by barbiturates and vitamin K. Coumarins are competitive inhibitors of vitamin K in the biosynthesis of prothrombin.
The coagulation cascade relies on the conversion of prothrombin to thrombin in a very important step. However, this conversion depends on the presence of 10 g-carboxyglutamic acid (GLA) residues in the N-terminus of prothrombin. The multiple Gla residues form a binding site for Ca2+. Under normal circumstances 10 glutamic acid (Glu) residues of prothrombin are converted to Gla residues in a post-translational modification.
This post-translation modification is catalyzed by an enzymes vitamin K reductase and vitamin K epoxide reductase. Vitamin K is a co-factor in this conversion reaction. Thus it cycles between a reduced form and an epoxide form. Because of their structural similarity with vitamin K coumarins are thought to bind the enzymes, vitamin K reductase and vitamin K epoxide reductase, without facilitating the conversion of Glu residues of prothrombin to Gla. Thus prothrombin cannot be acted upon by factor Xa.
The 1,3-indanediones have been known in the art to be anticoagulant since the 1940s. The onset and duration of action of anisindione are similar to those for coumarins. The chief disadvantage of indandiones is their side effects. Some patients are hypersensitive to it and develop a rash, pyrexia, and leukopenia.
Despite the overall benefits achieved, the currently used therapeutic anticoagulants are also a major source of mortality and morbidity, caused by limitations in efficacy and even more so by bleeding complications. In an effort to overcome these problems, a number of new agents have been developed. However, it appears that therapeutic anticoagulation inevitably comes with the inherent problem that increased efficiency is only achieved by an increase in bleeding complications. Targeting of anticoagulants to the clot may represent a means to break this fatal linkage. The fusion of anticoagulants to antibodies that are directed against clot-specific epitopes allows enrichment of the anticoagulants at the clot whereas the concentration of the anticoagulants in the circulating blood can be kept at a low level.
The success of clot targeting is dependent on the abundance and specificity of the epitope chosen as target. It has been previously demonstrated that fibrin, may be used for clot targeting. However fibrin or fibrin degradation products may circulate in the blood leading to mis-targeting of anticoagulants in the circulation.
A further problem in the art relates to the diagnosis of clotting disorders. It is accepted that many clotting disorders may be prevented or at least prevented from advancing to a more serious problem. It is therefore desirable for the clinician to have an indicator of early clotting disorders.
It is an aspect of the present invention to overcome or alleviate a problem of the prior art by providing an anticoagulant agent that is efficacious, yet does not result in extended clotting time. The present invention further provides methods and reagents for diagnosing a clotting-related disorder.
The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.